- Email: [email protected]
Studies on construction of a recombinant Eimeria tenella SO7 gene expressing Escherichia coli and its protective efficacy against homologous infection
Parasitology International 59 (2010) 517–523
Contents lists available at ScienceDirect
Parasitology International j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / p a r i n t
Studies on construction of a recombinant Eimeria tenella SO7 gene expressing Escherichia coli and its protective efficacy against homologous infection Guilian Yang a,1, Chunfeng Wang a,⁎,1, Fengqi Hao b, Dan Zhao a, Yunlong Zhang a, Yu Li c,⁎ a b c
College of Animal Science and Technology, Jilin Agricultural University, 2888 Xincheng Street, Changchun 130118, China College of Life Science and Technology, Changchun University of Science and Technology, 7989 Weixing Road, Changchun 130022, China Edible and Medical Fungi Engineering Research Center of Chinese Ministry of Education, Jilin Agricultural University, 2888 Xincheng Street, Changchun 130118, China
a r t i c l e
i n f o
Article history: Received 16 November 2009 Received in revised form 7 June 2010 Accepted 23 June 2010 Available online 1 July 2010 Keywords: Eimeria tenella SO7 gene Non-antibiotic Escherichia coli Protective effect
a b s t r a c t Eimeria spp. are the causative agents of coccidiosis, a major disease affecting the poultry industry. A recombinant non-antibiotic Escherichia coli that expresses the Eimeria tenella SO7 gene was constructed and its protective efficacy against homologous infection in chickens was determined. The three-day-old chickens were orally immunized with the recombinant non-antibiotic SO7 gene expressing E. coli and boosted two weeks later. Four weeks after the second immunization, the chickens were challenged with 5 × 104 homologous sporulated oocysts. The protective effects of the recombinant non-antibiotic E. coli were determined by measuring body weight change, mortality, histopathology, lesion scores, oocyst counts, the specific antibody response and the frequency of CD4+ and CD8+ lymphocytes in peripheral blood. The results showed that immunization with SO7 expressing E. coli resulted in significantly improved body weight gain, reduced lesion scores and oocyst shedding in immunized chickens compared to controls. Furthermore, administration of recombinant SO7 expressing E. coli leads to a significant increase in serum antibody, CD4+ and CD8+ T cells in peripheral blood of chickens. These results, therefore, suggest that the recombinant nonantibiotic E. coli that expresses the SO7 gene is able to effectively stimulate host protective immunity as evidenced by the induction of development of both humoral and cell-mediated immune responses against homologous challenge in chickens. © 2010 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Coccidiosis is one of the most important protozoan diseases affecting the poultry industry [1]. Eimeria tenella is considered to be one of the most pathogenic Eimeria organisms parasitizing the chicken. Large-scale control of coccidiosis in the poultry industry still relies largely on prophylactic chemotherapy, which is known to result in the development of drug resistance [2]. Infection of the host with Eimeria spp. leads to the development of strong antigen-specific protective immunity. Early studies provided evidence to support the feasibility of using vaccines for effective control of coccidiosis in the intensive poultry industry. A live vaccine was introduced in the USA a half century ago and its variants are still being used today. However, safety concerns still remain in using the live, non-attenuated vaccine strains. With the long-term aim of developing a safer recombinant
⁎ Corresponding authors. Wang, College of Animal Science and Technology, Jilin Agricultural University, 2888 Xincheng Street, Changchun 130118, China. Li is to be contacted at Edible and Medical Fungi Engineering Research Center of Chinese Ministry of Education, Jilin Agricultural University, 2888 Xincheng Street, Changchun 130118, China. Tel./fax: +86 431 84533425. E-mail addresses: [email protected] (C. Wang), [email protected] (Y. Li). 1 These authors contributed equally to this paper. 1383-5769/$ – see front matter © 2010 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.parint.2010.06.010
vaccine for the control of avian coccidiosis, an approach involving the expression of Eimeria vaccine antigens in live, attenuated viral or bacterial vectors has been considered [2]. Eimerian genes have been cloned and expressed in intestinal bacteria and viruses and the oral inoculation of such recombinant bacteria and viruses induced antigen-specific T and B cell responses [3–6]. The approach that uses live bacteria as a carrier to express and deliver the heterologous protective antigen genes shows great potential for large-scale control of infectious diseases in the livestock industry. The SO7 gene was screened from the E. tenella λgt11 cDNA library with rabbit antiserum raised against antigens extracted from sporulated oocysts [7]. The SO7 gene was inserted into the pJC264 vector and was expressed in E. coli as a fusion protein, CheY-SO7, which was purified and used to immunize chickens. Immunization with this recombinant protein not only could provide protection against homologous infection but also protected chickens from heterologous infection, suggesting that the SO7 gene may be a good candidate for use in recombinant vaccine development [8]. In order to construct a non-antibiotic recombinant vaccine against coccidiosis and to evaluate its protective potential against homologous infection, a food-grade shuttle vector named pMG36t (lacking any antibiotic resistance genes), which can transfer genes between E. coli and Lactobacillus by using the thymidylate synthase (thyA) gene as selective
518
G. Yang et al. / Parasitology International 59 (2010) 517–523
marker, was constructed. The SO7 gene was sub-cloned into the pMG36t vector resulting in creation of the recombinant vector plasmid pMG36tSO7. The pMG36t-SO7 was then transformed into a thyA gene mutant E. coli that was isolated from healthy chicken intestinal tract. The protective effects of immunization with pMG36t-SO7-E. coli were determined by measuring oocyst output, body weight change, lesion scores, serum antibody and lymphocyte proliferation responses. 2. Materials and methods 2.1. Chickens, parasites, bacteria and plasmids One-day-old specific-pathogen free (SPF) chickens were reared in a coccidian-free environment in wire cages. Feed and water were supplied ad libitum. The highly virulent E. tenella Beijing strain isolated from chickens in Beijing was used in this study. The oocysts were sporulated and purified according to methods described elsewhere [9]. E. coli χ13, a E. coli thyA gene mutant strain, was isolated from the intestinal tract of healthy chickens with the CaCl2 method [10,11]. The pMG36e vector plasmid was kindly provided by the Institute of Microbiology, Chinese Academy of Sciences, China. 2.2. Construction of non-antibiotic recombinant expression vector A DNA fragment containing the thyA gene was excised from the pMD18-thyA plasmid by digestion with NsiI and PstI. It was ligated to the NsiI and PstI digested pMG36e vector, which can transfer genes between E. coli and Lactobacillus species, after filling in the PstI sites with the Klenow enzyme. In this process, the erythromycin resistance gene present in pMG36e was replaced with the thyA fragment, thus generating a plasmid, pMG36t, that lacks any antibiotic resistance genes. A DNA fragment containing the SO7 gene was ligated into the XbaI and KpnI sites of pMG36t to produce the pMG36t-SO7 expression vector, which was verified by restriction digests and sequencing. The pMG36t-SO7 construction scheme is detailed in Fig. 1. 2.3. Identification of positive recombinant E. coli and stability of pMG36t-SO7 construct The pMG36t-SO7 vector was transformed into the recipient thyA mutant E. coli χ13 using the CaCl2 method [10]. The positive recombinant clone was identified on selective LB plate (without thymidylic acid), which was termed E. coli χ2. To determine the proportions of cells that can retain the Thy+ plasmids from each culture after 14 h of growth (approximately 50 generations), 10− 5 and 10− 6 dilutions were plated onto thymidylic acid supplemented LB plates (50 μg/ml). After overnight incubation, 100 clones of E. coli χ2 were picked and replated onto LB plates with and without thymidylic acid supplement (50 μg/ml). Colonies that grew on both types of LB plates were counted, and the percentage of clones retaining the plasmids was determined [5]. 2.4. Identification of SO7 protein expressed in E. coli χ2 The cultivation, transformation and induction of the expression of SO7 gene were carried out as described previously. Briefly, the E. coli χ2 was cultured in LB medium without thymidylic acid, and harvested at mid-log phase by centrifugation. After induction at 37 °C for 2 h, the bacterial pellets were resuspended in 1 ml of STE (100 mM NaCl, 10 mM Tris hydrochloride, 1 mM EDTA, pH 7.0), incubated at 37 °C for 15 min, and disrupted on ice with sonication. Protein concentration of the culture lysates was determined. For immunoblotting, proteins were separated by SDS-PAGE, and transferred to nitrocellulose membranes. The membranes were blocked with 5% skim milk in 100 mM Tris (0.9% NaCl and 0.1% Tween 20, pH 7.4), incubated with mouse antibody specific for SO7 (prepared by immunizing mouse with the SO7 protein, which
was obtained from the expressing E. coli prepared in this study), and followed by alkaline phosphatase-conjugated goat anti-mouse immunoglobulin G (Sigma). Immunoreactive bands were detected by addition of nitroblue tetrazolium (NBT)-5-bromo-4-chloro-3-indolylphosphate (BCIP) (Sigma). The reaction was stopped after 2 to 5 min by washing the membranes several times with large volumes of deionized water. 2.5. Immunization and challenge of chickens by E. tenella sporulated oocysts Three-day-old SPF chickens were randomly divided into four groups of 50 each. All chickens were deprived of food and water for 4 h before oral immunization. Each chicken in groups 1 and 2 was orally immunized with recombinant E. coli χ2 transformed pMG36t-SO7 and pMG36t plasmid, respectively (106 CFU). At the same time, the chickens in group 3 were orally administrated E. coli χ13 (106 CFU). Chickens in group 4 were orally administrated PBS as controls. Extreme care was taken to avoid accidental exposure of chickens to coccidians during this period and feces from chickens of each group were periodically examined for the presence of oocysts. A boosting immunization was carried out with the same dose at 2 weeks postimmunization (p.i.). 20 chickens from each group were euthanized to collect blood for ELISA and FACS at 1, 2, 3 and 4 weeks p.i. Two weeks after the boosting immunization, the other 30 chickens from each group were challenged with 5 × 104 E. tenella sporulated oocysts per chicken and the surveillance for coccidian oocysts was continued until day 7 post-challenge, and these chickens were euthanized at day 8 post-challenge. The experimental design and immunization protocol are outlined in Table 1. 2.6. Immune response induced by E. coli χ2 in chickens Whole blood from 20 chickens per group was collected randomly by cardiac puncture at 1, 2, 3, and 4 weeks p.i., and serum samples were collected. Anti-parasite (E. tenella) antibody responses were determined using ELISA as described previously [12,13]. Briefly, 96well plates were coated with Eimeria antigen (10 μg/ml, 100 μl/well in carbonate/bicarbonate buffer pH 9.6). After overnight incubation at 4 °C, the plates were washed (three times with PBS containing 0.05% Tween 20). The plates were blocked (PBS supplemented with 10% fetal calf serum) for 1 h at 37 °C. Diluted serum samples (1:300 in PBS containing 10% fetal calf serum, 0.05% Tween 20) were added in duplicate and incubated overnight at 4 °C. After washing, the plates were incubated with peroxidase conjugated rabbit anti-chicken IgG antibody (Sigma) for 2 h at 37 °C (100 μl/well at 1:500 dilution). After washing, peroxidase activity was detected by adding orthophenylene diamine (Sigma) (100 μl/well). The reaction was stopped by addition of 2 N H2SO4 (50 μl/well) after incubation for 15 min at 37 °C. The plates were read at 490 nm in an automated plate reader (Bio-Rad, Hercules, CA). In addition, the cell-mediated immunity induced by E. coli χ2 was evaluated by examining the change of CD4+ and CD8+ cells in peripheral blood. Blood sample (100 μl) was incubated with fluorescein-conjugated mouse anti-chicken CD4 antibody (0.5 μg/μl) and R-phycoerythrin-conjugated mouse anti-chicken CD8 antibody (0.5 μg/μl) (BD, San Jose, CA) for 40 min at room temperature in the dark. After two washes with PBS, cells were resuspended in a + fluorescence preservative fluid and the proportions of CD+ 4 and CD8 cells were analyzed by FACS on a FACScalibur (BD Bioscience, San Jose, CA). Dead cells and debris were excluded from analysis by gates set on forward and side angle light scatter. 2.7. Evaluation of the protective effect of E. coli χ2 against homologous infection To determine the protective effect of E. coli χ2, we monitored the body weight gains (BWG) and mortality, and examined oocysts
G. Yang et al. / Parasitology International 59 (2010) 517–523
519
Fig. 1. Construction of non-antibiotic expression vector pMG36t-SO7. The pMG36t-SO7 plasmid was constructed as described in the text.
output and cecal lesion in chickens treated with E. coli χ2. The initial and final body weights of the experimental chickens in each group were determined. Fecal samples from chickens of each group were collected between 5th and 9th days post-challenge. The oocyst output was determined using McMaster's counting technique. The efficacy of E. coli χ2 immunization was determined according to the reported method by Rose and Mockett [14]. Percentage protection = (the number of oocysts from control chickens − the number of oocysts from vaccinated chickens) / the number of oocysts from control chickens × 100%. The cecal tissue samples were collected and fixed with 10% neutral buffered formalin, embedded in paraffin, and stained
1
30
2
30
3 4
30 30
E. coli χ2 (pMG36t-SO7) E. coli χ2 (pMG36t) E. coli χ13 PBS
Secondary Oocysts challenge immunization (4 weeks p.i., (2 weeks p.i., 106 CFU) oocysts/chicken) E. coli χ2 (pMG36t-SO7) E. coli χ2 (pMG36t) E. coli χ13 PBS
2.8. Statistical analyses All results were analyzed by one-way ANOVA using Duncan's multiple range tests with SPSS 14.0 software. A p value of b0.05 was considered significant. 3. Results 3.1. Construction and stability of recombinant pMG36-SO7 vector and expression of SO7 protein in E. coli χ2
Table 1 Experimental design and immune program. Groups Number Primary of chickens immunization (106 CFU)
with hematoxylin and eosin (HE), 6 days after challenge with sporulated oocysts of E. tenella. The cecal lesion scores were determined according to the method of Johnson and Reid [15].
5 × 104 5 × 104 5 × 104 5 × 104
In order to construct the shuttle vector pMG36t lacking any antibiotic resistance genes, the erythromycin resistance gene was replaced by the thyA gene in the pMG36e vector. The pMG36t vector can transfer genes between E. coli and Lactobacillus species using the thyA gene as a selectable marker. The SO7 gene was sub-cloned into pMG36t to create the expression vector pMG36t-SO7, which was confirmed by restriction endonuclease digestion and sequencing (data not shown). SDS-PAGE and immunoblot analysis further showed that a specific band of about 26 kDa expressed in E. coli χ2
520
G. Yang et al. / Parasitology International 59 (2010) 517–523
(Fig. 2A) can be detected with antibodies against E. tenella (Fig. 2B). The results showed that the SO7 gene was expressed successfully in E. coli χ2. The stability of this pMG36t vector plasmid was demonstrated by its detection in the E. coli χ13 thyA− host strain after 50 or more generations of growth in the presence or absence of thymidylic acid (data not shown).
3.2. Immunization with recombinant E. coli χ2 (pMG36t-SO7) induces antibody responses The results of ELISA performed on serum samples from both immunized and control chickens are shown in Fig. 3. No obvious antibody response was detected in chickens immunized with E. coli χ2 (pMG36t-SO7) at 1 week p.i. However, a significant increase in mean absorbance values was detected in chickens immunized with E. coli χ2 (pMG36t-SO7) at the second week p.i. and remained high throughout the experimental period (weeks 2–4 p.i.), compared to the other groups. The induced humoral immune response level of chickens immunized with E. coli χ2 (pMG36t-SO7) was much higher than in those vaccinated with control vectors: E. coli χ2 (pMG36t) and E. coli χ13 (P b 0.05). There were no significant differences between antibody levels of chickens immunized with E. coli χ2 (pMG36t) and E. coli χ13 (P N 0.05). No antibody was detected in the control chickens.
3.3. Immunization with recombinant E. coli χ2 (pMG36t-SO7) increases peripheral CD4+ and CD8+ T lymphocyte cell frequency To further determine the systemic effect of immunization of E. coli χ2 (pMG36t-SO7), peripheral blood samples were collected and the frequencies of CD4+ and CD8+ T cells were measured using FACS. The results showed that treatment with recombinant E. coli χ2 (pMG36t-SO7) in chickens resulted in an increase in the frequency (and total numbers) of CD4+ and CD8+ cells in peripheral blood of chickens (Fig. 4). The frequency of both CD4+ and CD8+ T cells of chickens that received E. coli χ2 (pMG36t-SO7) was significantly higher than that detected in chickens from groups 2, 3 and 4 (P b 0.05). The observation that recombinant E. coli χ2 (pMG36t-SO7) was capable of inducing strong immune responses in chickens provided strong evidence to demonstrate the immunogenicity of recombinant E. coli χ2 (pMG36t-SO7), suggesting the potential of the recombinant E. coli χ2 (pMG36t-SO7) to induce strong protective immunity in chickens.
3.4. Demonstration of protective effects of E. coli χ2 expressing SO7 gene against E. tenella infection 3.4.1. Clinical symptom after challenge 5 days after challenge with E. tenella sporulated oocysts, chickens in group 2, 3 and control group showed clinical signs, such as anorexia, loss of weight, depression, hemorrhagic feces, and a high mortality rate (Table 2). We observed a 32.5%, 32% and 34% mortality rate in chickens from groups 2, 3 and 4, respectively. Massive concretionary blood was seen in the split cecum of the dead chickens. All clinical symptoms were typical of coccidiosis. In sharp contrast, chickens in group 1, which were immunized with recombinant E. coli χ2(pMG36t-SO7), did not show any of the symptoms described above, demonstrating that strong protection is induced in the immunized animals. Chickens that were immunized gained significantly higher body weight than those in groups 2, 3 and 4 (P b 0.05). No mortality was found in group 1. 3.4.2. Histopathologic analysis of cecum Microscopic examination showed that severe colonic inflammation developed in chickens infected with E. tenella, specifically in groups 2, 3 and 4 (control). In addition, in these groups, high numbers of schizonts and oocysts were observed in the cecal mucosa of chickens (Fig. 5). In contrast, no obvious change was observed in cecal sections of chickens in group 1 (immunized group). The lesion scores of group 1 was significantly lower (score: 1.1–1.6) than in other groups (2, 3 and 4) (P b 0.05). No significant differences were detected among groups 2, 3 and 4 (P N 0.05). 3.4.3. E. coli χ2 SO7 immunization results in reduced oocyst output We next examined whether E. coli χ2 SO7 immunization affected oocyst output by determining the OPG value. The results showed that oocyst output was significantly lower in group 1 (immunized group) compared to other groups (2, 3 and 4) (P b 0.05). There were no significant differences detected among other groups (2, 3 and 4) (P N 0.05). Taken together, these results demonstrate that the recombinant E. coli χ2 (pMG36t-SO7) is capable of inducing a strong protective immunity against E. tenella infection in chickens. 4. Discussion Coccidiosis is a major parasitic disease that can cause severe losses in poultry meat and egg production. It has been estimated that the coccidiosis induced economic losses are ranging from $800 million to $1.5 billion per year in poultry industry worldwide [16]. Development
Fig. 2. (A) SDS-PAGE analysis of the expression of E. tenella SO7 gene in recombinant E. coli χ2. The recombinant E. coli χ2 (pMG36t-SO7) was cultured in LB medium (without thymidine acid) and lysates analyzed on a 15% polyacrylamide gel and then stained with Coomassie blue. The result showed that there was a 26 kDa specific protein band in E. coli χ2 (pMG36t-SO7) compared with the control E. coli χ2 (pMG36t). Lane M: Low molecular weight protein marker; Lanes 1–6: Recombinant E. coli χ2 (pMG36t-SO7); Lane C: Recombinant E. coli χ2 (pMG36t) served as negative control. (B) The SO7 protein expressed in E. coli χ2 (pMG36t-SO7) was detected by Western blotting. Lane 7: E. coli χ2 (pMG36t-SO7); lane 8: E. coli χ2 (pMG36t) as the negative control.
G. Yang et al. / Parasitology International 59 (2010) 517–523
521
Table 2 Comparison of protective efficacy against E. tenella challenge in chickens immunized with different vaccine. Groups
Average BWG (g)
Lesion scores
OPG (107 oocysts/chicken)
Mortality (%)
Protection ratio (%)
1 2 3 4
325.7 ± 6.5A 210.6 ± 4.4B 211.8 ± 6.4B 208.0 ± 3.2B
1.31 ± 0.05A 3.48 ± 0.02B 3.50 ± 0.07B 3.52 ± 0.04B
5.81 ± 0.48A 18.51 ± 0.47B 18.12 ± 0.25B 18.03 ± 0.41B
0 32.5 32 34
67.7 – – –
All data were analyzed by SPSS 14.0 software and expressed as mean ± S.E. (n = 30). Values followed by the different capital letter are significantly different (P b 0.05).
Fig. 3. Antibody titers against the SO7 antigen in sera of chickens. Chickens were immunized i.g. with E. coli χ2 (pMG36t-SO7), E. coli χ2 (pMG36t), E. coli χ13 and PBS as control, and boosted 2 weeks later. Serum samples were collected from chickens at 1, 2, 3 and 4 weeks p.i. and the antibody titers were determined using ELISA. A490 of sera diluted 1:500 is shown. Each bar represents the mean OD (± S.E., n = 20), the asterisks indicate significantly increased serum antibody titers compared with the PBS control (P b 0.05).
of more effective vaccines against coccidiosis is needed [17]. In recent years live and attenuated vaccines have been used in prevention of coccidiosis. However, a disadvantage of this approach is the narrow margin of safety in using live, non-attenuated vaccines strains. In addition, large-scale manufacture of live attenuated vaccines is very
Fig. 4. (A) Changes of T lymphocyte in peripheral blood of immunized chickens. (A). + Changes of T CD+ 4 , (B) changes of CD8 T lymphocyte in peripheral blood of immunized chickens. Chickens were immunized as described in Fig. 3 legend. Blood samples were collected from chickens at 1, 2, 3 and 4 weeks p.i. and analyzed for the changes of CD+ 4 and CD+ 8 T lymphocytes using FACS. FACS analysis was performed on a FACScalibur (BD Bioscience, San Jose, CA). Each bar represents the mean percentage (± S.E., n = 20), the asterisks indicate significant difference (P b 0.05).
expensive [2]. The development of a new generation of vaccine, such as using recombinant techniques to express parasite antigen(s), may provide a safer, more effective and cost-effective approach for disease control. For a successful recombinant vaccine, an effective antigen delivery system is essential. The route of antigen administration and the antigen selected are also important determinants of success. It is now well accepted that the mucosal route of vaccination is the one that is most likely to induce strong immunity against Eimeria infection. The mucosal immune system is unique in its ability to develop tolerance to harmless antigens such as those derived from food and commensal bacteria [18]. However, when antigens such as Eimeria antigens are expressed and delivered in live, attenuated viral or bacterial vectors in the presence of adjuvants, a productive immunity can be induced. Several bacterial species such as E. coli, Salmonella, Listeria and Shigella have been successfully used to transfer eukaryotic expression plasmids into host cells [19–21]. In order to avoid using antibiotic resistance as a selection marker, which is prohibited by the FDA, pMG36t vector that complements the thyA mutation of E. coli host strain χ13 was constructed. The pMG36t vector is a shuttle vector that can transfer genes between E. coli and Lactobacillus. For expression of the protective antigen gene, E. coli host strain χ13 with a deletion of the thyA gene, which is essential for synthesis of the bacterial thymidylic acid, was used. The thyA deletion in host strains is complemented with antigen-bearing ThyA+ plasmids. The results of SDS-PAGE and Western blotting indicated that the E. tenella gene could be expressed in E. coli χ2 and the protein that is expressed in this system retains its immunogenecity. The successful construction of this shuttle vector lacking antibiotic resistance genes lays a foundation for future studies using the Lactobacillus expression system, which expresses and delivers the protective antigen genes of Eimeria effectively. In addition to the significance of identifying the best route for vaccine delivery, the selection of appropriate parasite antigens is equally important. A vaccine must contain antigen(s) from the parasite species that represent common antigenicity and may produce cross-immunity. Previously the SO7 gene was screened from the E. tenella λgt11 cDNA library [7]. A fusion protein CheY-SO7, delivered using intramuscular injection, induced protection against challenge with four different species of Eimeria [8]. In the current study, the SO7 gene from E. tenella was sub-cloned into the shuttle vector pMG36t to construct recombinant vector pMG36t-SO7. We showed that the SO7 protein was expressed successfully in E. coli as indicated by western blotting analysis. One of the characteristics of the immune response to Eimeria infection is the development of Th1-type and IFN-γ-mediated immunity with NK cells, cytotoxic CD8+ T cells and helper CD4+ T cells at the mucosal site of infection [22]. During infection, antibodies specific to Eimeria are generated and might be important for neutralizing Eimeria while it is in the extracellular stages of life cycle [22,23]. In the current study, we measured and compared the level of CD4+, CD8+ T cells in the peripheral blood and the antibody in the serum in chickens of different groups. We observed that the level of CD4+, CD8+ T cells in group 1 (vaccinated) was significantly higher than that detected in the other groups (groups 2, 3 and 4) (P b 0.05),
522
G. Yang et al. / Parasitology International 59 (2010) 517–523
Fig. 5. Histopathologic analysis of cecum in chickens challenged by E. tenella oocysts. The chickens were euthanised and cecal tissue samples were excised, fixed in 10% neutral buffered formalin and embedded in paraffin at seven days post-challenge with E. tenella sporulated oocysts. Tissue sections were stained with hematoxylin and eosin (HE). (A) Chickens were immunized with E. coli χ2 (pMG36t-SO7); (B) chickens were immunized with E. coli χ2 (pMG36t); (C) chickens were immunized with E. coli χ13; (D) chickens were treated with PBS as control. Magnification, × 400.
and that the level of antibody in group 1 was also higher than that in the other groups (groups 2, 3 and 4) (P b 0.05). These observations provide strong evidence to demonstrate that the SO7 protein expressed in recombinant E. coli χ2 could induce cell-mediated and humoral immunity in chickens. The results were further supported by the significant reduction in oocyst output and cecal pathological damage. To further demonstrate the protective efficacy of our newly established recombinant vaccine against E. tenella, we evaluated the BWG, a measurement that is considered to be the best index to measure the protective effect of a recombinant vaccine against coccidiosis [24]. BWG in group 1 was significantly higher than that in other groups (P b 0.05), indicating that vaccination with E. coli χ2 (pMG36t-SO7) resulted in prevention of body weight loss of chickens caused by E. tenella infection. The oocyst output and duration of shedding in group 1 were significantly lower than that of other groups (P b 0.05), which correlated with enhanced health status of chickens in immunized group without any clinical symptoms and mortality. Protection provided by immunization with E. coli χ2 (pMG36t-SO7) was further shown by the reduced pathologic change in the intestine. According to the OPG value of each group, the protection ratio of group 1 was 67.7%, which was similar to the results that were reported previously by Cranes et al. [8]. Our results, therefore, demonstrated that the recombinant E. coli that expresses the SO7 gene can induce protective immune responses and provide partial protection in chickens against homologous challenge. However, further investigations are needed to determine whether this recombinant vaccine can protect chickens that are challenged with heterologous species (E. acervulina, E. maxima and E. necatrix). In conclusion, the results demonstrate the successful construction of a vector that expresses E. tenella SO7 antigen using ThyA mutations of host strain E. coli χ13. Our data also showed that immunization of chickens with this vaccine strain induced the antigen-specific
immune responses, providing evidence to support the use of bacteria as vehicles in development of novel vaccines against E. tenella infection. Despite the fact that pMG36t was found to be a safe shuttle expression vector, our ongoing research focuses on optimizing vaccination conditions, such as dose of E. coli administered, timing of the primary and boosting immunizations. We are also attempting to improve the design of the E. coli strains and antigen delivery strategies in order to enhance the protective efficacy against E. tenella infection in the chicken industry. Acknowledgements This work was supported by a grant from the National Program for High Technology Research (2006AA10A205, 2007AA10Z322) and grants from the National Natural Science Foundation of China (NSFC, 30671573, 30700602 and 30870116). We gratefully acknowledge Binrui Xu for help with doing HE stain. References [1] Williams RB. A compartmentalised model for the estimation of the cost of coccidiosis to the world's chicken production industry. Int J Parasitol 1999;29: 1209–29. [2] McDonald V, Shirley MW. Past and future: vaccination against Eimeria. Parasitology 2009;136:1477–89. [3] Yang G, Li J, Zhang X, Zhao Q, Liu Q, Gong P. Eimeria tenella: construction of a recombinant fowlpox virus expressing rhomboid gene and its protective efficacy against homologous infection. Exp Parasitol 2008;119:30–6. [4] Pogonka T, Klotz C, Kovács F, Lucius R. A single dose of recombinant Salmonella typhimurium induces specific humoral immune responses against heterologous Eimeria tenella antigens in chicken. Int J Parasitol 2003;33:81–8. [5] Konjufca V, Wanda S-Y, Jenkins MC, Curtiss III R. A recombinant attenuated Salmonella enterica serovar Typhimurium vaccine encoding Eimeria acervulina antigen offers protection against E. acervulina challenge. Infect Immun 2006;74: 6785–96. [6] Konjufca V, Jenkins M, Wang S, Juarez-Rodriguez MD, Curtiss III R. Immunogenicity of recombinant attenuated Salmonella enterica serovar Typhimurium vaccine
G. Yang et al. / Parasitology International 59 (2010) 517–523
[7]
[8]
[9] [10] [11]
[12]
[13]
[14] [15]
strains carrying a gene that encodes Eimeria tenella antigen SO7. Infect Immun 2008;76:5745–53. Profous-Juchelka HLP, Turner M. Identification and characterization of cDNA clones encoding antigens of Eimeria tenella. Mol Biochem Parasitol 1988;30: 233–41. Crane MS, Goggin B, Pellegrino RM, Ravino OJ, Lange C, Karkhanis YD, et al. Crossprotection against four species of chicken coccidia with a single recombinant antigen. Infect Immun 1991;59:1271–7. Davis LR. Techniques. In: Hammond DM, Long PL, editors. The Coccidia. Baltimore: University Park Press; 1973. p. 411–58. Sambrook JAR, D.W.. Molecular Cloning: A Laboratory Manuel. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press; 2001. Wang C. Construction of expression-vector with non-antibiotics selection in Lactobacillus acidophilus and expression of SO7 gene of Eimeria tenella. College of Veterinary Medicine, 56. Beijing, China: China Agricultural University; 2001. Oz HS, Markham RJF, Bemrick WJ, Stromberg BE. Enzyme-linked immunosorbent assay and indirect haemagglutination techniques for measurement of antibody responses to Eimeria tenella in experimentally infected chickens. J Parasitol 1984;70:859–63. Constantinoiu CC, Molloy JB, Jorgensen WK, Coleman GT. Development and validation of an ELISA for detecting antibodies to Eimeria tenella in chickens. Vet Parasitol 2007;150:306–13. Rose ME, Mockett APA. Antibodies to coccidia: detection by the enzyme-linked immunosorbent assay (ELISA). Parasite Immunol 1983;5:479–89. Johnson J, Reid WM. Anticoccidial drugs: lesion scoring techniques in battery and floor-pen experiments with chickens. Exp Parasitol 1970;28:30–6.
523
[16] Shirley MW, Smith AL, Tomley FM, Baker JR, RMaDR. The biology of avian Eimeria with an emphasis on their control by vaccination. Advances in Parasitology. Academic Press; 2005. p. 285–330. [17] Vermeulen AN, Schaap DC, Schetters TPM. Control of coccidiosis in chickens by vaccination. Vet Parasitol 2001;100:13–20. [18] MacDonald TT, Monteleone G. Immunity, inflammation, and allergy in the gut. Science 2005;307:1920–5. [19] Gentschev I, Dietrich G, Spreng S, Kolb-Mäurer A, Brinkmann V, Grode L, et al. Recombinant attenuated bacteria for the delivery of subunit vaccines. Vaccine 2001;19:2621–8. [20] Liljeqvist S, Ståhl S. Production of recombinant subunit vaccines: protein immunogens, live delivery systems and nucleic acid vaccines. J Biotechnol 1999;73:1–33. [21] Bermudes D, Low B, Pawelek J. Tumor-targeted Salmonella. Highly selective delivery vectors in. Cancer Gene Ther 2000:57–63. [22] Lillehoj HS, Lillehoj EP. Avian coccidiosis. A review of acquired intestinal immunity and vaccination strategies. Avian Dis 2000;44:408–25. [23] Lillehoj HS. Effects of immunosuppression on avian coccidiosis: cyclosporin A but not hormonal bursectomy abrogates host protective immunity. Infect Immun 1987;55:1616–21. [24] Kopko SH, Martin DS, Barta JR. Responses of chickens to a recombinant refractile body antigen of Eimeria tenella administered using various immunizing strategies. Poult Sci 2000;79:336–42.
Contents lists available at ScienceDirect
Parasitology International j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / p a r i n t
Studies on construction of a recombinant Eimeria tenella SO7 gene expressing Escherichia coli and its protective efficacy against homologous infection Guilian Yang a,1, Chunfeng Wang a,⁎,1, Fengqi Hao b, Dan Zhao a, Yunlong Zhang a, Yu Li c,⁎ a b c
College of Animal Science and Technology, Jilin Agricultural University, 2888 Xincheng Street, Changchun 130118, China College of Life Science and Technology, Changchun University of Science and Technology, 7989 Weixing Road, Changchun 130022, China Edible and Medical Fungi Engineering Research Center of Chinese Ministry of Education, Jilin Agricultural University, 2888 Xincheng Street, Changchun 130118, China
a r t i c l e
i n f o
Article history: Received 16 November 2009 Received in revised form 7 June 2010 Accepted 23 June 2010 Available online 1 July 2010 Keywords: Eimeria tenella SO7 gene Non-antibiotic Escherichia coli Protective effect
a b s t r a c t Eimeria spp. are the causative agents of coccidiosis, a major disease affecting the poultry industry. A recombinant non-antibiotic Escherichia coli that expresses the Eimeria tenella SO7 gene was constructed and its protective efficacy against homologous infection in chickens was determined. The three-day-old chickens were orally immunized with the recombinant non-antibiotic SO7 gene expressing E. coli and boosted two weeks later. Four weeks after the second immunization, the chickens were challenged with 5 × 104 homologous sporulated oocysts. The protective effects of the recombinant non-antibiotic E. coli were determined by measuring body weight change, mortality, histopathology, lesion scores, oocyst counts, the specific antibody response and the frequency of CD4+ and CD8+ lymphocytes in peripheral blood. The results showed that immunization with SO7 expressing E. coli resulted in significantly improved body weight gain, reduced lesion scores and oocyst shedding in immunized chickens compared to controls. Furthermore, administration of recombinant SO7 expressing E. coli leads to a significant increase in serum antibody, CD4+ and CD8+ T cells in peripheral blood of chickens. These results, therefore, suggest that the recombinant nonantibiotic E. coli that expresses the SO7 gene is able to effectively stimulate host protective immunity as evidenced by the induction of development of both humoral and cell-mediated immune responses against homologous challenge in chickens. © 2010 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Coccidiosis is one of the most important protozoan diseases affecting the poultry industry [1]. Eimeria tenella is considered to be one of the most pathogenic Eimeria organisms parasitizing the chicken. Large-scale control of coccidiosis in the poultry industry still relies largely on prophylactic chemotherapy, which is known to result in the development of drug resistance [2]. Infection of the host with Eimeria spp. leads to the development of strong antigen-specific protective immunity. Early studies provided evidence to support the feasibility of using vaccines for effective control of coccidiosis in the intensive poultry industry. A live vaccine was introduced in the USA a half century ago and its variants are still being used today. However, safety concerns still remain in using the live, non-attenuated vaccine strains. With the long-term aim of developing a safer recombinant
⁎ Corresponding authors. Wang, College of Animal Science and Technology, Jilin Agricultural University, 2888 Xincheng Street, Changchun 130118, China. Li is to be contacted at Edible and Medical Fungi Engineering Research Center of Chinese Ministry of Education, Jilin Agricultural University, 2888 Xincheng Street, Changchun 130118, China. Tel./fax: +86 431 84533425. E-mail addresses: [email protected] (C. Wang), [email protected] (Y. Li). 1 These authors contributed equally to this paper. 1383-5769/$ – see front matter © 2010 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.parint.2010.06.010
vaccine for the control of avian coccidiosis, an approach involving the expression of Eimeria vaccine antigens in live, attenuated viral or bacterial vectors has been considered [2]. Eimerian genes have been cloned and expressed in intestinal bacteria and viruses and the oral inoculation of such recombinant bacteria and viruses induced antigen-specific T and B cell responses [3–6]. The approach that uses live bacteria as a carrier to express and deliver the heterologous protective antigen genes shows great potential for large-scale control of infectious diseases in the livestock industry. The SO7 gene was screened from the E. tenella λgt11 cDNA library with rabbit antiserum raised against antigens extracted from sporulated oocysts [7]. The SO7 gene was inserted into the pJC264 vector and was expressed in E. coli as a fusion protein, CheY-SO7, which was purified and used to immunize chickens. Immunization with this recombinant protein not only could provide protection against homologous infection but also protected chickens from heterologous infection, suggesting that the SO7 gene may be a good candidate for use in recombinant vaccine development [8]. In order to construct a non-antibiotic recombinant vaccine against coccidiosis and to evaluate its protective potential against homologous infection, a food-grade shuttle vector named pMG36t (lacking any antibiotic resistance genes), which can transfer genes between E. coli and Lactobacillus by using the thymidylate synthase (thyA) gene as selective
518
G. Yang et al. / Parasitology International 59 (2010) 517–523
marker, was constructed. The SO7 gene was sub-cloned into the pMG36t vector resulting in creation of the recombinant vector plasmid pMG36tSO7. The pMG36t-SO7 was then transformed into a thyA gene mutant E. coli that was isolated from healthy chicken intestinal tract. The protective effects of immunization with pMG36t-SO7-E. coli were determined by measuring oocyst output, body weight change, lesion scores, serum antibody and lymphocyte proliferation responses. 2. Materials and methods 2.1. Chickens, parasites, bacteria and plasmids One-day-old specific-pathogen free (SPF) chickens were reared in a coccidian-free environment in wire cages. Feed and water were supplied ad libitum. The highly virulent E. tenella Beijing strain isolated from chickens in Beijing was used in this study. The oocysts were sporulated and purified according to methods described elsewhere [9]. E. coli χ13, a E. coli thyA gene mutant strain, was isolated from the intestinal tract of healthy chickens with the CaCl2 method [10,11]. The pMG36e vector plasmid was kindly provided by the Institute of Microbiology, Chinese Academy of Sciences, China. 2.2. Construction of non-antibiotic recombinant expression vector A DNA fragment containing the thyA gene was excised from the pMD18-thyA plasmid by digestion with NsiI and PstI. It was ligated to the NsiI and PstI digested pMG36e vector, which can transfer genes between E. coli and Lactobacillus species, after filling in the PstI sites with the Klenow enzyme. In this process, the erythromycin resistance gene present in pMG36e was replaced with the thyA fragment, thus generating a plasmid, pMG36t, that lacks any antibiotic resistance genes. A DNA fragment containing the SO7 gene was ligated into the XbaI and KpnI sites of pMG36t to produce the pMG36t-SO7 expression vector, which was verified by restriction digests and sequencing. The pMG36t-SO7 construction scheme is detailed in Fig. 1. 2.3. Identification of positive recombinant E. coli and stability of pMG36t-SO7 construct The pMG36t-SO7 vector was transformed into the recipient thyA mutant E. coli χ13 using the CaCl2 method [10]. The positive recombinant clone was identified on selective LB plate (without thymidylic acid), which was termed E. coli χ2. To determine the proportions of cells that can retain the Thy+ plasmids from each culture after 14 h of growth (approximately 50 generations), 10− 5 and 10− 6 dilutions were plated onto thymidylic acid supplemented LB plates (50 μg/ml). After overnight incubation, 100 clones of E. coli χ2 were picked and replated onto LB plates with and without thymidylic acid supplement (50 μg/ml). Colonies that grew on both types of LB plates were counted, and the percentage of clones retaining the plasmids was determined [5]. 2.4. Identification of SO7 protein expressed in E. coli χ2 The cultivation, transformation and induction of the expression of SO7 gene were carried out as described previously. Briefly, the E. coli χ2 was cultured in LB medium without thymidylic acid, and harvested at mid-log phase by centrifugation. After induction at 37 °C for 2 h, the bacterial pellets were resuspended in 1 ml of STE (100 mM NaCl, 10 mM Tris hydrochloride, 1 mM EDTA, pH 7.0), incubated at 37 °C for 15 min, and disrupted on ice with sonication. Protein concentration of the culture lysates was determined. For immunoblotting, proteins were separated by SDS-PAGE, and transferred to nitrocellulose membranes. The membranes were blocked with 5% skim milk in 100 mM Tris (0.9% NaCl and 0.1% Tween 20, pH 7.4), incubated with mouse antibody specific for SO7 (prepared by immunizing mouse with the SO7 protein, which
was obtained from the expressing E. coli prepared in this study), and followed by alkaline phosphatase-conjugated goat anti-mouse immunoglobulin G (Sigma). Immunoreactive bands were detected by addition of nitroblue tetrazolium (NBT)-5-bromo-4-chloro-3-indolylphosphate (BCIP) (Sigma). The reaction was stopped after 2 to 5 min by washing the membranes several times with large volumes of deionized water. 2.5. Immunization and challenge of chickens by E. tenella sporulated oocysts Three-day-old SPF chickens were randomly divided into four groups of 50 each. All chickens were deprived of food and water for 4 h before oral immunization. Each chicken in groups 1 and 2 was orally immunized with recombinant E. coli χ2 transformed pMG36t-SO7 and pMG36t plasmid, respectively (106 CFU). At the same time, the chickens in group 3 were orally administrated E. coli χ13 (106 CFU). Chickens in group 4 were orally administrated PBS as controls. Extreme care was taken to avoid accidental exposure of chickens to coccidians during this period and feces from chickens of each group were periodically examined for the presence of oocysts. A boosting immunization was carried out with the same dose at 2 weeks postimmunization (p.i.). 20 chickens from each group were euthanized to collect blood for ELISA and FACS at 1, 2, 3 and 4 weeks p.i. Two weeks after the boosting immunization, the other 30 chickens from each group were challenged with 5 × 104 E. tenella sporulated oocysts per chicken and the surveillance for coccidian oocysts was continued until day 7 post-challenge, and these chickens were euthanized at day 8 post-challenge. The experimental design and immunization protocol are outlined in Table 1. 2.6. Immune response induced by E. coli χ2 in chickens Whole blood from 20 chickens per group was collected randomly by cardiac puncture at 1, 2, 3, and 4 weeks p.i., and serum samples were collected. Anti-parasite (E. tenella) antibody responses were determined using ELISA as described previously [12,13]. Briefly, 96well plates were coated with Eimeria antigen (10 μg/ml, 100 μl/well in carbonate/bicarbonate buffer pH 9.6). After overnight incubation at 4 °C, the plates were washed (three times with PBS containing 0.05% Tween 20). The plates were blocked (PBS supplemented with 10% fetal calf serum) for 1 h at 37 °C. Diluted serum samples (1:300 in PBS containing 10% fetal calf serum, 0.05% Tween 20) were added in duplicate and incubated overnight at 4 °C. After washing, the plates were incubated with peroxidase conjugated rabbit anti-chicken IgG antibody (Sigma) for 2 h at 37 °C (100 μl/well at 1:500 dilution). After washing, peroxidase activity was detected by adding orthophenylene diamine (Sigma) (100 μl/well). The reaction was stopped by addition of 2 N H2SO4 (50 μl/well) after incubation for 15 min at 37 °C. The plates were read at 490 nm in an automated plate reader (Bio-Rad, Hercules, CA). In addition, the cell-mediated immunity induced by E. coli χ2 was evaluated by examining the change of CD4+ and CD8+ cells in peripheral blood. Blood sample (100 μl) was incubated with fluorescein-conjugated mouse anti-chicken CD4 antibody (0.5 μg/μl) and R-phycoerythrin-conjugated mouse anti-chicken CD8 antibody (0.5 μg/μl) (BD, San Jose, CA) for 40 min at room temperature in the dark. After two washes with PBS, cells were resuspended in a + fluorescence preservative fluid and the proportions of CD+ 4 and CD8 cells were analyzed by FACS on a FACScalibur (BD Bioscience, San Jose, CA). Dead cells and debris were excluded from analysis by gates set on forward and side angle light scatter. 2.7. Evaluation of the protective effect of E. coli χ2 against homologous infection To determine the protective effect of E. coli χ2, we monitored the body weight gains (BWG) and mortality, and examined oocysts
G. Yang et al. / Parasitology International 59 (2010) 517–523
519
Fig. 1. Construction of non-antibiotic expression vector pMG36t-SO7. The pMG36t-SO7 plasmid was constructed as described in the text.
output and cecal lesion in chickens treated with E. coli χ2. The initial and final body weights of the experimental chickens in each group were determined. Fecal samples from chickens of each group were collected between 5th and 9th days post-challenge. The oocyst output was determined using McMaster's counting technique. The efficacy of E. coli χ2 immunization was determined according to the reported method by Rose and Mockett [14]. Percentage protection = (the number of oocysts from control chickens − the number of oocysts from vaccinated chickens) / the number of oocysts from control chickens × 100%. The cecal tissue samples were collected and fixed with 10% neutral buffered formalin, embedded in paraffin, and stained
1
30
2
30
3 4
30 30
E. coli χ2 (pMG36t-SO7) E. coli χ2 (pMG36t) E. coli χ13 PBS
Secondary Oocysts challenge immunization (4 weeks p.i., (2 weeks p.i., 106 CFU) oocysts/chicken) E. coli χ2 (pMG36t-SO7) E. coli χ2 (pMG36t) E. coli χ13 PBS
2.8. Statistical analyses All results were analyzed by one-way ANOVA using Duncan's multiple range tests with SPSS 14.0 software. A p value of b0.05 was considered significant. 3. Results 3.1. Construction and stability of recombinant pMG36-SO7 vector and expression of SO7 protein in E. coli χ2
Table 1 Experimental design and immune program. Groups Number Primary of chickens immunization (106 CFU)
with hematoxylin and eosin (HE), 6 days after challenge with sporulated oocysts of E. tenella. The cecal lesion scores were determined according to the method of Johnson and Reid [15].
5 × 104 5 × 104 5 × 104 5 × 104
In order to construct the shuttle vector pMG36t lacking any antibiotic resistance genes, the erythromycin resistance gene was replaced by the thyA gene in the pMG36e vector. The pMG36t vector can transfer genes between E. coli and Lactobacillus species using the thyA gene as a selectable marker. The SO7 gene was sub-cloned into pMG36t to create the expression vector pMG36t-SO7, which was confirmed by restriction endonuclease digestion and sequencing (data not shown). SDS-PAGE and immunoblot analysis further showed that a specific band of about 26 kDa expressed in E. coli χ2
520
G. Yang et al. / Parasitology International 59 (2010) 517–523
(Fig. 2A) can be detected with antibodies against E. tenella (Fig. 2B). The results showed that the SO7 gene was expressed successfully in E. coli χ2. The stability of this pMG36t vector plasmid was demonstrated by its detection in the E. coli χ13 thyA− host strain after 50 or more generations of growth in the presence or absence of thymidylic acid (data not shown).
3.2. Immunization with recombinant E. coli χ2 (pMG36t-SO7) induces antibody responses The results of ELISA performed on serum samples from both immunized and control chickens are shown in Fig. 3. No obvious antibody response was detected in chickens immunized with E. coli χ2 (pMG36t-SO7) at 1 week p.i. However, a significant increase in mean absorbance values was detected in chickens immunized with E. coli χ2 (pMG36t-SO7) at the second week p.i. and remained high throughout the experimental period (weeks 2–4 p.i.), compared to the other groups. The induced humoral immune response level of chickens immunized with E. coli χ2 (pMG36t-SO7) was much higher than in those vaccinated with control vectors: E. coli χ2 (pMG36t) and E. coli χ13 (P b 0.05). There were no significant differences between antibody levels of chickens immunized with E. coli χ2 (pMG36t) and E. coli χ13 (P N 0.05). No antibody was detected in the control chickens.
3.3. Immunization with recombinant E. coli χ2 (pMG36t-SO7) increases peripheral CD4+ and CD8+ T lymphocyte cell frequency To further determine the systemic effect of immunization of E. coli χ2 (pMG36t-SO7), peripheral blood samples were collected and the frequencies of CD4+ and CD8+ T cells were measured using FACS. The results showed that treatment with recombinant E. coli χ2 (pMG36t-SO7) in chickens resulted in an increase in the frequency (and total numbers) of CD4+ and CD8+ cells in peripheral blood of chickens (Fig. 4). The frequency of both CD4+ and CD8+ T cells of chickens that received E. coli χ2 (pMG36t-SO7) was significantly higher than that detected in chickens from groups 2, 3 and 4 (P b 0.05). The observation that recombinant E. coli χ2 (pMG36t-SO7) was capable of inducing strong immune responses in chickens provided strong evidence to demonstrate the immunogenicity of recombinant E. coli χ2 (pMG36t-SO7), suggesting the potential of the recombinant E. coli χ2 (pMG36t-SO7) to induce strong protective immunity in chickens.
3.4. Demonstration of protective effects of E. coli χ2 expressing SO7 gene against E. tenella infection 3.4.1. Clinical symptom after challenge 5 days after challenge with E. tenella sporulated oocysts, chickens in group 2, 3 and control group showed clinical signs, such as anorexia, loss of weight, depression, hemorrhagic feces, and a high mortality rate (Table 2). We observed a 32.5%, 32% and 34% mortality rate in chickens from groups 2, 3 and 4, respectively. Massive concretionary blood was seen in the split cecum of the dead chickens. All clinical symptoms were typical of coccidiosis. In sharp contrast, chickens in group 1, which were immunized with recombinant E. coli χ2(pMG36t-SO7), did not show any of the symptoms described above, demonstrating that strong protection is induced in the immunized animals. Chickens that were immunized gained significantly higher body weight than those in groups 2, 3 and 4 (P b 0.05). No mortality was found in group 1. 3.4.2. Histopathologic analysis of cecum Microscopic examination showed that severe colonic inflammation developed in chickens infected with E. tenella, specifically in groups 2, 3 and 4 (control). In addition, in these groups, high numbers of schizonts and oocysts were observed in the cecal mucosa of chickens (Fig. 5). In contrast, no obvious change was observed in cecal sections of chickens in group 1 (immunized group). The lesion scores of group 1 was significantly lower (score: 1.1–1.6) than in other groups (2, 3 and 4) (P b 0.05). No significant differences were detected among groups 2, 3 and 4 (P N 0.05). 3.4.3. E. coli χ2 SO7 immunization results in reduced oocyst output We next examined whether E. coli χ2 SO7 immunization affected oocyst output by determining the OPG value. The results showed that oocyst output was significantly lower in group 1 (immunized group) compared to other groups (2, 3 and 4) (P b 0.05). There were no significant differences detected among other groups (2, 3 and 4) (P N 0.05). Taken together, these results demonstrate that the recombinant E. coli χ2 (pMG36t-SO7) is capable of inducing a strong protective immunity against E. tenella infection in chickens. 4. Discussion Coccidiosis is a major parasitic disease that can cause severe losses in poultry meat and egg production. It has been estimated that the coccidiosis induced economic losses are ranging from $800 million to $1.5 billion per year in poultry industry worldwide [16]. Development
Fig. 2. (A) SDS-PAGE analysis of the expression of E. tenella SO7 gene in recombinant E. coli χ2. The recombinant E. coli χ2 (pMG36t-SO7) was cultured in LB medium (without thymidine acid) and lysates analyzed on a 15% polyacrylamide gel and then stained with Coomassie blue. The result showed that there was a 26 kDa specific protein band in E. coli χ2 (pMG36t-SO7) compared with the control E. coli χ2 (pMG36t). Lane M: Low molecular weight protein marker; Lanes 1–6: Recombinant E. coli χ2 (pMG36t-SO7); Lane C: Recombinant E. coli χ2 (pMG36t) served as negative control. (B) The SO7 protein expressed in E. coli χ2 (pMG36t-SO7) was detected by Western blotting. Lane 7: E. coli χ2 (pMG36t-SO7); lane 8: E. coli χ2 (pMG36t) as the negative control.
G. Yang et al. / Parasitology International 59 (2010) 517–523
521
Table 2 Comparison of protective efficacy against E. tenella challenge in chickens immunized with different vaccine. Groups
Average BWG (g)
Lesion scores
OPG (107 oocysts/chicken)
Mortality (%)
Protection ratio (%)
1 2 3 4
325.7 ± 6.5A 210.6 ± 4.4B 211.8 ± 6.4B 208.0 ± 3.2B
1.31 ± 0.05A 3.48 ± 0.02B 3.50 ± 0.07B 3.52 ± 0.04B
5.81 ± 0.48A 18.51 ± 0.47B 18.12 ± 0.25B 18.03 ± 0.41B
0 32.5 32 34
67.7 – – –
All data were analyzed by SPSS 14.0 software and expressed as mean ± S.E. (n = 30). Values followed by the different capital letter are significantly different (P b 0.05).
Fig. 3. Antibody titers against the SO7 antigen in sera of chickens. Chickens were immunized i.g. with E. coli χ2 (pMG36t-SO7), E. coli χ2 (pMG36t), E. coli χ13 and PBS as control, and boosted 2 weeks later. Serum samples were collected from chickens at 1, 2, 3 and 4 weeks p.i. and the antibody titers were determined using ELISA. A490 of sera diluted 1:500 is shown. Each bar represents the mean OD (± S.E., n = 20), the asterisks indicate significantly increased serum antibody titers compared with the PBS control (P b 0.05).
of more effective vaccines against coccidiosis is needed [17]. In recent years live and attenuated vaccines have been used in prevention of coccidiosis. However, a disadvantage of this approach is the narrow margin of safety in using live, non-attenuated vaccines strains. In addition, large-scale manufacture of live attenuated vaccines is very
Fig. 4. (A) Changes of T lymphocyte in peripheral blood of immunized chickens. (A). + Changes of T CD+ 4 , (B) changes of CD8 T lymphocyte in peripheral blood of immunized chickens. Chickens were immunized as described in Fig. 3 legend. Blood samples were collected from chickens at 1, 2, 3 and 4 weeks p.i. and analyzed for the changes of CD+ 4 and CD+ 8 T lymphocytes using FACS. FACS analysis was performed on a FACScalibur (BD Bioscience, San Jose, CA). Each bar represents the mean percentage (± S.E., n = 20), the asterisks indicate significant difference (P b 0.05).
expensive [2]. The development of a new generation of vaccine, such as using recombinant techniques to express parasite antigen(s), may provide a safer, more effective and cost-effective approach for disease control. For a successful recombinant vaccine, an effective antigen delivery system is essential. The route of antigen administration and the antigen selected are also important determinants of success. It is now well accepted that the mucosal route of vaccination is the one that is most likely to induce strong immunity against Eimeria infection. The mucosal immune system is unique in its ability to develop tolerance to harmless antigens such as those derived from food and commensal bacteria [18]. However, when antigens such as Eimeria antigens are expressed and delivered in live, attenuated viral or bacterial vectors in the presence of adjuvants, a productive immunity can be induced. Several bacterial species such as E. coli, Salmonella, Listeria and Shigella have been successfully used to transfer eukaryotic expression plasmids into host cells [19–21]. In order to avoid using antibiotic resistance as a selection marker, which is prohibited by the FDA, pMG36t vector that complements the thyA mutation of E. coli host strain χ13 was constructed. The pMG36t vector is a shuttle vector that can transfer genes between E. coli and Lactobacillus. For expression of the protective antigen gene, E. coli host strain χ13 with a deletion of the thyA gene, which is essential for synthesis of the bacterial thymidylic acid, was used. The thyA deletion in host strains is complemented with antigen-bearing ThyA+ plasmids. The results of SDS-PAGE and Western blotting indicated that the E. tenella gene could be expressed in E. coli χ2 and the protein that is expressed in this system retains its immunogenecity. The successful construction of this shuttle vector lacking antibiotic resistance genes lays a foundation for future studies using the Lactobacillus expression system, which expresses and delivers the protective antigen genes of Eimeria effectively. In addition to the significance of identifying the best route for vaccine delivery, the selection of appropriate parasite antigens is equally important. A vaccine must contain antigen(s) from the parasite species that represent common antigenicity and may produce cross-immunity. Previously the SO7 gene was screened from the E. tenella λgt11 cDNA library [7]. A fusion protein CheY-SO7, delivered using intramuscular injection, induced protection against challenge with four different species of Eimeria [8]. In the current study, the SO7 gene from E. tenella was sub-cloned into the shuttle vector pMG36t to construct recombinant vector pMG36t-SO7. We showed that the SO7 protein was expressed successfully in E. coli as indicated by western blotting analysis. One of the characteristics of the immune response to Eimeria infection is the development of Th1-type and IFN-γ-mediated immunity with NK cells, cytotoxic CD8+ T cells and helper CD4+ T cells at the mucosal site of infection [22]. During infection, antibodies specific to Eimeria are generated and might be important for neutralizing Eimeria while it is in the extracellular stages of life cycle [22,23]. In the current study, we measured and compared the level of CD4+, CD8+ T cells in the peripheral blood and the antibody in the serum in chickens of different groups. We observed that the level of CD4+, CD8+ T cells in group 1 (vaccinated) was significantly higher than that detected in the other groups (groups 2, 3 and 4) (P b 0.05),
522
G. Yang et al. / Parasitology International 59 (2010) 517–523
Fig. 5. Histopathologic analysis of cecum in chickens challenged by E. tenella oocysts. The chickens were euthanised and cecal tissue samples were excised, fixed in 10% neutral buffered formalin and embedded in paraffin at seven days post-challenge with E. tenella sporulated oocysts. Tissue sections were stained with hematoxylin and eosin (HE). (A) Chickens were immunized with E. coli χ2 (pMG36t-SO7); (B) chickens were immunized with E. coli χ2 (pMG36t); (C) chickens were immunized with E. coli χ13; (D) chickens were treated with PBS as control. Magnification, × 400.
and that the level of antibody in group 1 was also higher than that in the other groups (groups 2, 3 and 4) (P b 0.05). These observations provide strong evidence to demonstrate that the SO7 protein expressed in recombinant E. coli χ2 could induce cell-mediated and humoral immunity in chickens. The results were further supported by the significant reduction in oocyst output and cecal pathological damage. To further demonstrate the protective efficacy of our newly established recombinant vaccine against E. tenella, we evaluated the BWG, a measurement that is considered to be the best index to measure the protective effect of a recombinant vaccine against coccidiosis [24]. BWG in group 1 was significantly higher than that in other groups (P b 0.05), indicating that vaccination with E. coli χ2 (pMG36t-SO7) resulted in prevention of body weight loss of chickens caused by E. tenella infection. The oocyst output and duration of shedding in group 1 were significantly lower than that of other groups (P b 0.05), which correlated with enhanced health status of chickens in immunized group without any clinical symptoms and mortality. Protection provided by immunization with E. coli χ2 (pMG36t-SO7) was further shown by the reduced pathologic change in the intestine. According to the OPG value of each group, the protection ratio of group 1 was 67.7%, which was similar to the results that were reported previously by Cranes et al. [8]. Our results, therefore, demonstrated that the recombinant E. coli that expresses the SO7 gene can induce protective immune responses and provide partial protection in chickens against homologous challenge. However, further investigations are needed to determine whether this recombinant vaccine can protect chickens that are challenged with heterologous species (E. acervulina, E. maxima and E. necatrix). In conclusion, the results demonstrate the successful construction of a vector that expresses E. tenella SO7 antigen using ThyA mutations of host strain E. coli χ13. Our data also showed that immunization of chickens with this vaccine strain induced the antigen-specific
immune responses, providing evidence to support the use of bacteria as vehicles in development of novel vaccines against E. tenella infection. Despite the fact that pMG36t was found to be a safe shuttle expression vector, our ongoing research focuses on optimizing vaccination conditions, such as dose of E. coli administered, timing of the primary and boosting immunizations. We are also attempting to improve the design of the E. coli strains and antigen delivery strategies in order to enhance the protective efficacy against E. tenella infection in the chicken industry. Acknowledgements This work was supported by a grant from the National Program for High Technology Research (2006AA10A205, 2007AA10Z322) and grants from the National Natural Science Foundation of China (NSFC, 30671573, 30700602 and 30870116). We gratefully acknowledge Binrui Xu for help with doing HE stain. References [1] Williams RB. A compartmentalised model for the estimation of the cost of coccidiosis to the world's chicken production industry. Int J Parasitol 1999;29: 1209–29. [2] McDonald V, Shirley MW. Past and future: vaccination against Eimeria. Parasitology 2009;136:1477–89. [3] Yang G, Li J, Zhang X, Zhao Q, Liu Q, Gong P. Eimeria tenella: construction of a recombinant fowlpox virus expressing rhomboid gene and its protective efficacy against homologous infection. Exp Parasitol 2008;119:30–6. [4] Pogonka T, Klotz C, Kovács F, Lucius R. A single dose of recombinant Salmonella typhimurium induces specific humoral immune responses against heterologous Eimeria tenella antigens in chicken. Int J Parasitol 2003;33:81–8. [5] Konjufca V, Wanda S-Y, Jenkins MC, Curtiss III R. A recombinant attenuated Salmonella enterica serovar Typhimurium vaccine encoding Eimeria acervulina antigen offers protection against E. acervulina challenge. Infect Immun 2006;74: 6785–96. [6] Konjufca V, Jenkins M, Wang S, Juarez-Rodriguez MD, Curtiss III R. Immunogenicity of recombinant attenuated Salmonella enterica serovar Typhimurium vaccine
G. Yang et al. / Parasitology International 59 (2010) 517–523
[7]
[8]
[9] [10] [11]
[12]
[13]
[14] [15]
strains carrying a gene that encodes Eimeria tenella antigen SO7. Infect Immun 2008;76:5745–53. Profous-Juchelka HLP, Turner M. Identification and characterization of cDNA clones encoding antigens of Eimeria tenella. Mol Biochem Parasitol 1988;30: 233–41. Crane MS, Goggin B, Pellegrino RM, Ravino OJ, Lange C, Karkhanis YD, et al. Crossprotection against four species of chicken coccidia with a single recombinant antigen. Infect Immun 1991;59:1271–7. Davis LR. Techniques. In: Hammond DM, Long PL, editors. The Coccidia. Baltimore: University Park Press; 1973. p. 411–58. Sambrook JAR, D.W.. Molecular Cloning: A Laboratory Manuel. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press; 2001. Wang C. Construction of expression-vector with non-antibiotics selection in Lactobacillus acidophilus and expression of SO7 gene of Eimeria tenella. College of Veterinary Medicine, 56. Beijing, China: China Agricultural University; 2001. Oz HS, Markham RJF, Bemrick WJ, Stromberg BE. Enzyme-linked immunosorbent assay and indirect haemagglutination techniques for measurement of antibody responses to Eimeria tenella in experimentally infected chickens. J Parasitol 1984;70:859–63. Constantinoiu CC, Molloy JB, Jorgensen WK, Coleman GT. Development and validation of an ELISA for detecting antibodies to Eimeria tenella in chickens. Vet Parasitol 2007;150:306–13. Rose ME, Mockett APA. Antibodies to coccidia: detection by the enzyme-linked immunosorbent assay (ELISA). Parasite Immunol 1983;5:479–89. Johnson J, Reid WM. Anticoccidial drugs: lesion scoring techniques in battery and floor-pen experiments with chickens. Exp Parasitol 1970;28:30–6.
523
[16] Shirley MW, Smith AL, Tomley FM, Baker JR, RMaDR. The biology of avian Eimeria with an emphasis on their control by vaccination. Advances in Parasitology. Academic Press; 2005. p. 285–330. [17] Vermeulen AN, Schaap DC, Schetters TPM. Control of coccidiosis in chickens by vaccination. Vet Parasitol 2001;100:13–20. [18] MacDonald TT, Monteleone G. Immunity, inflammation, and allergy in the gut. Science 2005;307:1920–5. [19] Gentschev I, Dietrich G, Spreng S, Kolb-Mäurer A, Brinkmann V, Grode L, et al. Recombinant attenuated bacteria for the delivery of subunit vaccines. Vaccine 2001;19:2621–8. [20] Liljeqvist S, Ståhl S. Production of recombinant subunit vaccines: protein immunogens, live delivery systems and nucleic acid vaccines. J Biotechnol 1999;73:1–33. [21] Bermudes D, Low B, Pawelek J. Tumor-targeted Salmonella. Highly selective delivery vectors in. Cancer Gene Ther 2000:57–63. [22] Lillehoj HS, Lillehoj EP. Avian coccidiosis. A review of acquired intestinal immunity and vaccination strategies. Avian Dis 2000;44:408–25. [23] Lillehoj HS. Effects of immunosuppression on avian coccidiosis: cyclosporin A but not hormonal bursectomy abrogates host protective immunity. Infect Immun 1987;55:1616–21. [24] Kopko SH, Martin DS, Barta JR. Responses of chickens to a recombinant refractile body antigen of Eimeria tenella administered using various immunizing strategies. Poult Sci 2000;79:336–42.